Transcriptional regulation of human CYP3A4 basal expression by CCAAT enhancer-binding protein alpha and hepatocyte nuclear factor-3 gamma.

Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of more than 50% of currently used therapeutic drugs, yet the mechanisms that control CYP3A4 basal expression in liver are poorly understood. Several putative binding sites for CCAAT/enhancer-binding protein (C/EBP) and hepatic nuclear factor 3 (HNF-3) were found by computer analysis in CYP3A4 promoter. The use of reporter gene assays, electrophoretic mobility shift assays, and site-directed mutagenesis revealed that one proximal and two distal C/EBP alpha binding sites are essential sites for the trans-activation of CYP3A4 promoter. No trans-activation was found in similar reporter gene experiments with a HNF-3 gamma expression vector. The relevance of these findings was further explored in the more complex DNA/chromatin structure within endogenous CYP3A4 gene. Using appropriate adenoviral expression vectors, we found that both hepatic and nonhepatic cells overexpressing C/EBP alpha had increased CYP3A4 mRNA levels, but no effect was observed when HNF-3 gamma was overexpressed. In contrast, overexpression of HNF-3 gamma simultaneously with C/EBP alpha resulted in a greater activation of the CYP3A4 gene. This cooperative effect was hepatic-specific and also occurred in CYP3A5 and CYP3A7 genes. To investigate the mechanism for HNF-3 gamma action, we studied its binding to CYP3A4 promoter and the effect of the deacetylase inhibitor trichostatin A. HNF-3 gamma was able to bind CYP3A4 promoter at a distal position, near the most distal C/EBP alpha binding site. Trichostatin A increased C/EBP alpha effect but abolished HNF-3 gamma cooperative action. These findings revealed that C/EBP alpha and HNF-3 gamma cooperatively regulate CYP3A4 expression in hepatic cells by a mechanism that probably involves chromatin remodeling.

Neuroprotective agent chlomethiazole attenuates c-fos, c-jun, and AP-1 activation through inhibition of p38 MAP kinase.

Recent evidence suggests that stress-activated protein kinases expressed in glial cells have very important roles during cerebral ischemia. The neuroprotective agent chlomethiazole, which is known to enhance the conductance at the GABA(A) receptor complex, is presently in clinical trials for the treatment of severe stroke. Here the authors suggested that chlormethiazole has anti-inflammatory properties because it potently and selectively inhibited p38 mitogen-activated protein (MAP) kinase in primary cortical glial cultures. The inhibition of p38 MAP kinase resulted in the attenuation of the induction of c-fos and c-jun mRNA and AP-1 DNA binding by lipopolysaccharide (LPS). In addition, chlomethiazole inhibited the activation of an AP-1-dependent luciferase reporter plasmid in SK-N-MC human neuroblastoma cells in response to glutamate. Chlomethiazole inhibited the p38 MAP kinase activity as revealed by the decrease in the LPS-induced phosphorylation of the substrates ATF-2 and hsp27, whereas the phosphorylation status of the p38 MAP kinase itself was unaffected. Interestingly, chlomethiazole exhibited an IC(50) of approximately 2 micromol/L for inhibition of c-fos mRNA expression, indicating 25 to 75 times higher potency than reported EC(50) values for enhancing GABA(A) chloride currents. The results indicated a novel mechanism of action of chlomethiazole, and provided support for a distinctive role of p38 MAP kinase in cerebral ischemia.

Contribution of MAP kinase pathways to the activation of ATF-2 in human neuroblastoma cells.

Activated Transcription Factor-2 (ATF-2) is important during development of and during injury to the brain. Both Jun N-terminal Kinases (JNKs) and p38 Mitogen-Activated Protein Kinases (p38MAPKs) may phosphorylate ATF-2, but the contribution of these two pathways in cells has never been investigated. We have assayed endogenous p38MAPK activity in SK-N-MC and SH-SY5Y human neuroblastoma cells for activation of a GAL4/ATF-2 fusionprotein, by means of titrations of transfected expression plasmids and by using the p38MAPK inhibitor SB203580. It was found that basal activation of ATF-2 was independent of p38MAPK and that whereas MAPK kinase-3 (MKK3) was a weak inducer of ATF-2 activation, it was a potent activator of the stress activated transcription factor CHOP. In contrast, ATF-2 was very potently activated by the JNK pathway activator MAPK kinase kinase-1 (MEKK1). Thus, kinases downstream of MEKK1 appear relevant, but it is unlikely that p38MAPKs contribute quantitatively to activation of ATF-2 in these cells.

Interindividual differences in hepatic expression of CYP3A4: relationship to genetic polymorphism in the 5'-upstream regulatory region.

Cytochrome P450 3A4, the most abundant P450 form in human liver, exhibits a very broad substrate specificity and is of great importance for drug metabolism. The interindividual difference in the hepatic expression of CYP3A4 is considerable. In order to investigate possible genetic factor(s) causing this variation, the rate of 6beta-hydroxylation of testosterone in human liver microsomes prepared from 46 different human liver samples was determined and the 5'upstream region (+10 to -490 bp) was sequenced from genomic DNA isolated from 39 of these livers. We found a 31-fold variation of the testosterone hydroxylase activity between the samples. However, a very high sequence homology between the CYP3A4 5'-upstream regions sequenced from the 78 different alleles was found. In fact, only three variant nucleotide exchanges were identified, all causing a -290 A-->G mutation (CYP3A4-V) in a so called nifedipine specific element (NFSE). The importance of this element and the polymorphism was evaluated by gel shift analysis. Competition experiments revealed that the binding of nuclear proteins, although having lower affinity to the CYP3A4-V form of the element, was unspecific in nature. In accordance, no influence of this polymorphism was seen on the microsomal testosterone hydroxylase activity in vitro. It is concluded that the promoter region of CYP3A4 is highly conserved, the only polymorphism being in the NFSE, which however does not influence the enzyme expression in liver to a significant degree. This casts doubt of a previously described relationship between the CYP3A4-V allele and cancer in the prostate and leukaemia.

Expression and distribution of cytochrome P450 enzymes in male rat kidney: effects of ethanol, acetone and dietary conditions.

Ethanol, acetone, diet and starvation, known modulators of the hepatic cytochrome P450 (CYP)-dependent microsomal monooxygenase system, were assessed for their effects on cytochrome P450 isozyme content and monooxygenase activities in the male rat kidney. In acute experiments, rats were either treated with acetone, fasted or given a combination of the two treatments. Acetone treatment alone induced CYP2E1-dependent p-nitrophenol hydroxylase activity in kidney microsomes by 8-fold. This was accompanied by a 6-fold increase in CYP2E1 apoprotein as determined by Western blot analysis. There was, however, no significant increase in steady-state levels of CYP2E1 mRNA as measured by Northern blot analysis. Starvation also induced CYP2E1 apoprotein in the kidney and, as has been reported previously in the liver, had a synergistic inductive effect with acetone. CYP2B and CYP3A apoproteins were also induced by acetone, starvation and starvation/acetone combinations in the kidney. Immunohistochemical analysis revealed localization of CYP2E1 and CYP2B principally in the cortex associated with tubular cells. This distribution was maintained upon starvation/acetone treatment. Two induction experiments were performed in which the ethanol was administered as part of a system of total enteral nutrition (TEN). A short-term study was conducted in which ethanol was administered for 8 days in two liquid diets of different composition, and a chronic experiment was performed in which ethanol was administered for 35 days. A diet-independent 6-fold increase in CYP2E1 apoprotein was observed in the short-term experiment. Expression of CYP3A and CYP2A cross-reactive apoproteins in kidney microsomes appeared to be affected by alterations in diet but, were unaffected by ethanol treatment. In the chronic 35-day ethanol exposure experiment, CYP2E1 apoprotein was also elevated 6-fold and this was found to be accompanied by a significant 3-fold increase in CYP2E1 mRNA. In the same study, no ethanol effects were apparent on expression of CYP2B and CYP3A apoproteins. Thus, acetone induced a variety of renal cytochrome P450 forms in addition to CYP2E1, while ethanol appeared to be a much more specific renal CYP2E1 inducer. Furthermore, as reported in the liver, acetone and ethanol appeared to induce CYP2E1 in the kidney by different mechanisms.

Expression, catalytic activity, and inducibility of cytochrome P450 2E1 (CYP2E1) in the rat central nervous system.

Cytochrome P450 2E1 (CYP2E1) metabolizes several neuroactive substrates, including exogenous compounds such as anesthetics, organic solvents, and muscle relaxants as well as endogenous substrates such as arachidonic acid. CYP2E1 and its mRNA were found to be expressed in the rat hippocampus, where the enzyme was localized mainly to the microsomal fraction. Chlorzoxazone (CZN), a CYP2E1 substrate, was 6-hydroxylated in hippocampal homogenates with a K(m) of 25.5 microM and a Vmax of 0.22 pmol/mg/min. CYP2E1 was also expressed in vitro in cortical glial cultures, where CYP2E1 mRNA levels were found to be 1,000-fold lower than in rat liver. Exposure of cortical glial cultures to 25 or 100 mM ethanol for 24 h caused a fourfold and sixfold increase, respectively, in the rate of CYP2E1-dependent 6-hydroxylation of CZN. After a continuous exposure to 100 mM ethanol for 48 or 72 h, however, the hydroxylation rate was down-regulated. Chlormethiazole, a potent inhibitor of hepatic CYP2E1 transcription, inhibited the ethanol-dependent induction of CYP2E1 by 50%. In vivo, acute ethanol treatment of rats (24 h, 3 g/kg) resulted in a 1.8-fold increase in the rate of CZN 6-hydroxylation in hippocampal homogenates. It is concluded that CYP2E1 is expressed and catalytically active in the rat CNS, and that CYP2E1 can be induced by a relatively low concentration of ethanol in cortical glial cultures. It is suggested that CYP2E1 substrates may be metabolically activated in situ in the CNS.

Induction of cytochrome P450 2E1 expression in rat and gerbil astrocytes by inflammatory factors and ischemic injury.

Hepatic cytochromes P450 are known to be down-regulated by cytokines, lipopolysaccharide, Gram-positive bacteria, and viruses. Little is known, however, about the regulation by inflammation of cytochromes P450 in other tissues. We have found that lipopolysaccharide and interleukin-1 beta stimulate the expression of catalytically active CYP2E1 (but not CYP1A1 or CYP2B) up to 7-fold in rat brain primary cortical glial cultures. The induction reached a maximum after 24 hr and was accompanied by an increase in CYP2E1 mRNA. Chlormethiazole, a specific inhibitor of hepatic CYP2E1 transcription, completely inhibited the induction of CYP2E1 at the mRNA and enzyme levels. Immunofluorescence studies showed CYP2E1 to be expressed in a subset of astrocytes in the lipopolysaccharide-stimulated cortical glial cultures. Using a model of global ischemic injury in the gerbil, we found CYP2E1 to be induced in vivo in astrocytes in the inflammatory phase, 1-3 weeks after the lesion. Likewise, CYP2E1 was induced in the rat cortex 1 week after a focal ischemic injury. Our results suggest tissue-specific regulation of CYP2E1 by inflammatory factors and that CYP2E1 may play a role in astrocytes during inflammation in the brain.

Regional distribution of ethanol-inducible cytochrome P450 IIE1 in the rat central nervous system.

A specific form of cytochrome P450, P450 IIE1, active in ethanol oxidation, is known to be induced about 10-fold in rat liver following ethanol treatment. This isozyme of P450 participates effectively in the metabolic activation of precarcinogens, such as N-dimethylnitrosamines, and of solvents such as carbon tetrachloride and benzene. In the present investigation, two different polyclonal antisera against P450 IIE1 were used in order to map the regional distribution of this P450 form in the rat central nervous system. The presence of P450 IIE1 in various brain regions was confirmed by Western blot analysis. The P450 IIE1-immunoreactivity was heterogeneously distributed between brain areas. Neuronal cell bodies and glial cells of presumed astroglial as well as oligodendroglial identity contained immunoreactivity. All fiber tracts harbored P450 IIE1-immunoreactive glial cells as did the ependymal lining of the ventricular wall as well as small and large vessels throughout the brain. P450 IIE1-immunoreactive glial cells were present in all areas of the neocortex, in the olfactory bulb, in the piriform cortex and in several different thalamic nuclei. In the cerebellum, P450 IIE-immunoreactivity was found in all cell layers and was exclusively localized to glial cells and their processes. Staining of blood vessels was prominent in the white matter where P450 IIE1-immunoreactive glial cells were seen to have end-feet on the vessels. A subgroup of pyramidal cells of the frontal cortex showed strong P450 IIE1-immunoreactivity, as did a component of the olfactory nerve which innervates the accessory bulb. In the hippocampal region, the pyramidal cells of all subfields were P450 IIE1-immunoreactive. Some polymorphic cells of the hilus and subfield CA stained intensely with the P450 IIE1 antibodies. A high density of P450 IIE1-immunoreactivity was detected throughout the striatal complex. The immunoreactivity was localized to neuronal cell bodies as well as the neurophil. Fibers of the nigrostriatal system were strongly P450 IIE1-immunoreactive. Mechanical lesions of this pathway showed an accumulation of P450 IIE1-immunoreactivity proximal to the lesion relative to the striatum and a depletion in the reticular part of the substantia nigra, suggesting that the antigen may be transported from the striatum to the substantia nigra. In the brain stem a high density of P450 IIE-immunoreactive neurons was detected in the substantia nigra, the pontine nucleus, lateral superior olive and the nucleus of the trigeminal nerve and facial nucleus. A great number of large- to medium-sized immunoreactive neurons were situated in the central gray and in the reticular formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytochrome P-450 and oxygen toxicity. Oxygen-dependent induction of ethanol-inducible cytochrome P-450 (IIE1) in rat liver and lung.

The ethanol-inducible form of cytochrome P-450 (P-450IIE1) has previously been shown to exhibit an unusually high rate of oxidase activity with the subsequent formation of reactive oxygen species, e.g., hydrogen peroxide, and to be the main contributor of microsomal oxidase activity in liver microsomes from acetone-treated rats [Ekström & Ingelman-Sundberg (1989) Biochem. Pharmacol. (in press)]. The results here presented indicate that oxygen exposure of rats causes an about 4-fold induction of P-450IIE1 in rat liver and lung microsomes. The induction in liver was not accompanied by any measurable increase in the P-450IIE1 mRNA levels, but the enhanced amount of P-450IIE1 accounted for 60% of the net 50% increase in the level of hepatic P-450 as determined spectrophotometrically. The induction of P-450IIE1 was maximal after 60 h of O2 exposure, and concomitant increases in the rates of liver microsomal CCl4-dependent lipid peroxidation, O2 consumption, NADPH oxidation, O2- formation, H2O2 production, and NADPH-dependent microsomal lipid peroxidation were seen. Liver microsomes from oxygen-treated rats had very similar properties to those of microsomes isolated from acetone-treated rats with respect to the P-450IIE1 content and catalytic properties, but different from those of thyroxine-treated animals. Treatment of rats with the P-450IIE1 inducer acetone in combination with oxygen exposure caused a potentiation of the NADPH-dependent liver and lung microsomal lipid peroxidation and decreased the survival time of the rats. The results reached indicate a role for cytochrome P-450 and, in particular, for cytochrome P-450IIE1 in oxygen-mediated tissue toxicity.